Correspondence/findings

Bcl11b is a transcription factor that is “monoallelically” inactivated in ~10 % of human T cell acute lymphoblastic leukemia (T-ALL) [1–3] and several murine T-ALL models, including ATM−/− thymic lymphomas [4–6]. Both human and murine T-ALLs retain at least one intact copy of Bcl11b, indicating that Bcl11b is a bona fide haploinsufficient tumor-suppressor gene. Complete loss of the Bcl11b gene abrogates T cell development and gain of NK cell phenotype, revealing a critical role of Bcl11b in T cell lineage commitment [7–10]. Conditional inactivation of both copies of Bcl11b in double-positive (DP) T cells leads to overproduction of innate CD8+ single-positive (SP) T cells [11] and compromises T-regulatory cell function [12]. Yet, hemizygous loss of Bcl11b has no measurable impact on T cell function [7–10] and the mechanism by which it promotes T-ALL remains elusive.

To address this, we characterized Bcl11b+/−ATM−/− mice [6, 13]. ATM kinase is a master regulator of the DNA damage responses [14]. ATM−/− mice routinely succumb to immature T cell lymphomas sharing molecular features with human T-ALL. Of the ATM−/− thymic lymphomas, 80 % hemizygously deleted Bcl11b as a result of non-reciprocal t(12;14) translocation [6, 15]. Conditional inactivation of Bcl11b in T cells via LckCre eliminates recurrent t(12;14) translocations from ATM−/− thymic lymphomas, suggesting Bcl11b as the target of the large chromosome 12 deletion [16]. ATM−/−Bcl11b+/− mice were born at expected frequency (Additional file 1: Figure S1A). Thymocyte development and peripheral T and B cell repertoire in young (4 week) ATM−/−Bcl11b+/− mice were indistinguishable from that of ATM−/− mice, displaying reduced surface TCRβ/CD3ε expression in DP cells and a partial blockade at the DP to SP transition characteristic for ATM-deficiency [6, 17] (Additional file 1: Figure S1B).

While we initially expected Bcl11b-deficiency to accelerate ATM-deficient thymic lymphomas based on its frequent inactivation in T-ALLs, hemizygous-deletion of Bcl11b prevented lethal thymic lymphoma in ~50 % ATM−/− mice and only 2/11 ATM−/−Bcl11b+/− mice developed overt thymic lymphomas (Fig. 1a). The median survival of ATM−/−Bcl11b+/− mice was significantly longer than that of ATM−/− controls (167 vs. 106 days, p < 0.01) (Fig. 1a). Analyses of 3- and 10-month-old ATM−/−Bcl11b+/− mice revealed clonal expansion of immature (surfaceTCRβlow) thymocytes in 3-month, but not 10-month-old ATM−/−Bcl11b+/− mice (Fig. 1b, c). T cell lymphomas from 3-month-old ATM−/−Bcl11b+/− mice retained both WT and null alleles of Bcl11b, consistent with the lack-of-LOH in T-ALL (Fig. 1d). Despite the clonal expansion in 3-month-old ATM−/−Bcl11b+/− mice, most thymic lymphomas failed to progress to lethal disease (Additional file 1: Figure S1C), suggesting that heterozygous Bcl11b-deficiency suppresses the progression, but not the initiation of ATM−/− thymic lymphomas.

Fig. 1
figure 1

Heterozygous loss of Bcl11b suppresses the progression, but not the initiation of ATM-deficient thymic lymphomas. a Thymic lymphoma-free survival of ATM−/− and ATM−/−Bcl11b+/− mice. Median survival of ATM−/− and ATM−/−Bcl11b+/− cohorts was 106 and 169 days, respectively. p value for log-ranking test is 0.0061. b Representative flow cytometry analyses of the thymus from control and ATM−/−Bcl11b+/− mice at 3 or 10 months of age. c Southern blot analyses of EcoRI digested genomic DNA from kidney (K), thymus (T), enlarged submandibular lymph nodes (L), or spleen (S) from ATM−/−Bcl11b+/− mice probed with TCRβ probes (Jβ1.6 or 2.7), TCRδ constant region (Cδ) or chromosome 14 amplification region (Amp) [6]. d Southern blot analyses of Bcl11b locus on KpnI digested genomic DNA with Bcl11b probe [13]

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Notably, almost all ATM−/−Bcl11b+/− mice developed variable degrees of splenomegaly, excessive submandibular lymph node (LN) enlargement that manifested to fatal airway obstruction (non-lymphoma-related death), and dermal inflammation commencing at 2–3 months of age (Fig. 2a). In contrast to normal lymphocyte profiles in 3-month-old ATM+/+(+/−)Bcl11b+/− mice (Additional file 1: Figure S2A and S2B), histologic analyses consistently revealed a lymphocyte-mediated inflammatory/immune disorder in ATM−/−Bcl11b+/− mice characterized by marked plasmacytosis with reactive germinal centers (B220+IgM+ Bcl6+CD138− B cells) in submandibular LN, reactive follicular hyperplasia of the white pulp and increased extramedullary hematopoiesis in the red pulp in the spleen (Fig. 2b and Additional file 1: Figure S2C), and acute and chronic dermal inflammation in the skin. While a cell-autonomous function of Bcl11b deletion on epidermal integrity cannot be ruled out [13], the splenic and LN changes noted raised the possibility of an autoimmune disorder, which could have contributed to the lack of tumor progression in ATM−/−Bcl11b+/− mice. Correspondingly, 10-month tumor-free ATM−/−Bcl11b+/− mice accumulated activated CD8+ T cell in PB (Fig. 2c).

Fig. 2
figure 2

Heterozygous loss of Bcl11b induces an inflammatory/immune response in ATM−/− mice. a Representative pictures of enlarged submandibular lymph nodes, dermatitis, and splenomegaly in ATM−/−Bcl11b+/− mice. b Histopathologic analysis of the submandibular lymph nodes and spleen in ATM−/−Bcl11b+/− mice. c Flow cytometry analysis shows significant enrichment of CD8+ SP T cells in the peripheral blood, but not the spleen of 10-month-old ATM−/−Bcl11b+/− mice

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Our data suggest that in an ATM-deficient background, heterozygous Bcl11b deficiency tilts immune homeostasis and limits the expansion, but not the initiation of ATM-deficient thymic lymphomas. Notably, homozygous Bcl11b deletion suppressed melanoma in murine models [18]. Given the role of Bcl11b in T cell lineage commitment, CD8+ T cell development, and T-reg function, our data suggest that heterozygous Bcl11b deficiency can modulate anti-tumor immune response despite the lack of measurable T cell development defects in Bcl11b+/− mice [7–10]. This role of Bcl11b in immune modulation and tumor suppression might explain the discrepancies between Bcl11b status (mutation, deletion, and downregulation) and T-ALL prognosis in different studies [1–3, 19]. It also suggests that Bcl11b is likely lost later during T-ALL development, as early deletion likely causes autoimmune dysfunction, analogous to TNFAIP3 (A20) in DLBCL [20].